傳染性華氏囊病毒次病毒顆粒與宿主細胞間之吸附特性分析

Abstract

傳染性華氏囊病毒(IBDV)是對幼雞隻具有高度傳染性的病毒，會造成養雞業者經濟上重大的損失。根據先前研究結果推測IBDV的外鞘(capsid)蛋白VP2是與病毒接受體結合並且具有宿主細胞辨識的功能。在透過昆蟲細胞表現系統單獨表現VP2蛋白時，VP2會自我組裝成一粒徑大小為25 nm的次病毒顆粒(SVP)。本研究目的是要藉由VP2蛋白所構成的次病毒顆粒探討其與IBDV宿主細胞的接受體之間的特性。首先發現到VP2會抑制病毒感染雞胚纖維母細胞，並且間接的影響病毒的複製，故推測說SVP會與病毒互相競爭接受體，為了要了解SVP與細胞之間的關系，以Vero與DF1作為IBDV的宿主細胞，被用來探討VP2與宿主細胞間的關係及特性。首先，發現SVP會吸附於Vero及DF1，再透過螢光顯微鏡的觀察，得知被FITC標定的SVP能吸附於宿主細胞Vero及DF1，之後由雷射共軛焦顯微鏡觀察SVP於細胞的分佈，觀測到SVP除了會吸附於細胞的表面外，並且還會進入到細胞質當中。在SVP與細胞吸附的特性分析方面，經由流式細胞儀的分析，發現吸附於細胞的SVP會隨著SVP濃度增加而提高的現象，並且還有飽和的趨勢，故可證明是專一性的吸附。更進一步地，再經由與IBDV之中和性單株抗體作用，能有效的抑制SVP與Vero、DF1之間的吸附，再度的指出SVP具有與IBDV 接受體(receptor)交互作用(interaction)的可能並且證實其在IBDV感染細胞的過程中伴演了重要的角色，IBDV感染時是以VP2蛋白作為媒介。

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. The outer capsid protein VP2 of IBDV has been suggested to play an important role in virus binding and cell recognition. VP2 can form a particle called subvirus particle (SVP) of 25 nm in diameter, when it was expressed in insect cells. This study attempts to identify the cellular receptors of IBDV susceptible cell lines using SVP. Inhibition infection assay with VP2-formed SVPs supports that SVPs competition with IBDV virons to the attachment of CEF and partially inhibit the replication of IBDV in CEF. Then, DF1 and Vero cells were used for study as host cell lines. Binding of VP2 particles to either DF1 or Vero cells was observed using various biochemical assays. The localization of the VP2 particles on Vero and DF1 cells was also confirmed by immunofluorescence microscope. The binding of the VP2-formed SVPs to Vero and DF1 cell surfaces was specific and occurred in a dose-dependent manner. Furthermore, the neutralizing monoclonal antibody against IBDV inhibits the attachment of SVPs particles to Vero and DF1 cells. The results suggest that the attachment of IBDV to susceptible cell is mediated by VP2.